1. Field of the Invention
The present invention relates to methods for the preparation of protein-containing polymeric materials, such as enzyme-containing polymeric materials. The present invention also relates to protein-containing polymeric materials and use of the materials, for example, as catalytic particles, in self-cleaning/non-fouling paints and coatings, as highly active and stable biocatalysts, in chemical/biochemical sensing and in medical applications including implants and in controlled drug release, immobilization, and/or stabilization of therapeutic proteins. The present invention encompasses biotechnological inventions, including biotechnological products and processes.
2. Background
Proteins, such as enzymes, are often immobilized to a support material in practical applications of biocatalysis. Numerous technologies are available for enzyme immobilization and include adsorption to a porous or nonporous support, covalent attachment to such a support, or entrapment in a solid or gelatinous support matrix. Although these approaches have served the biotechnology industry well over the years, several drawbacks have become evident. Such drawbacks include heterogeneity of enzyme loading onto the support, leakage or desorption of the biocatalyst from the support, and inactivation of the enzyme during immobilization procedures.
One approach to avoid these problems is to generate an extremely close association between the support and the biocatalyst. For example, immobilization of enzymes in hydrophilic or water-soluble polymers via polymerization in aqueous solution has been proposed and is described in the art. Such approaches have been used to prepare enzyme-containing hydrogels and other gel-like materials. Unfortunately, most of these materials are limited by the need to use highly water-soluble monomers or hydrophilic monomers, due to the solubility of enzymes that is generally limited to water and other polar solvents. For example, U.S. Pat. No. 4,727,030 to Fumihiro et al., describes the preparation of porous polyvinyl alcohol gel containing an immobilized enzyme. U.S. Pat. No. 4,371,612 describes immobilization of an enzyme via use of cross-linked microporous acrylonitrile polymers. U.S. Pat. No. 3,985,616 describes immobilization of an enzyme with gelatinized-starch-polyacrylonitrile graft polymers.
A few techniques have also been proposed for immobilization of an enzyme in organic media. U.S. Pat. No. 5,482,996 describes a protein immobilization process via covalent bonding in organic solvents. According to this patent, there is a need to modify the enzyme chemically by a modifier to dissolve the enzyme into organic media, which can alter the activity of the enzyme. As described below, such modification is not needed in the present invention. Also, the afore-mentioned modifier must be carefully controlled to be soluble in both aqueous and organic solutions and also possess a polymerizable functional groups for polymerization purpose. A typical example is acrylated polyethylene glycol, which is difficult and expensive to prepare. Another disadvantage of this process is that such modified enzymes usually show low solubility in organic solvents, thereby limiting the enzyme loading to about 0.02% by weight in the final polymer products. See Z. Yang, D. Williams, and A. J. Russell, J. Am. Chem. Soc., 1995, vol. 117, 4843. The solubilized enzyme of this process also shows lower activity as compared to the technology of the present invention. See V. M. Pardkar and J. S. Dordick, J. Am. Chem. Soc., 1994, vol. 116, 5009, and C. Pina, D. Clark, H. Blanch, and I. G. Gonegani, Biotechnology Techniques, 1989, vol. 3, 333.
Ito et al. (Biotechnol. Prog. 1993, 9, 128-130) describes another method of immobilization using organic solvents. Namely, Ito describes grafting enzymes with various hydrophobic vinyl polymers (e.g., polystyrene) in organic solvents by first coupling the enzyme with azobis (4-cyanovaleric acid) (ACV) in aqueous solution, followed by polymerization in organic solvents. However, the ACV-coupled enzyme is not soluble in the organic solvent, thus the chemical incorporation between the enzyme and polymer is significantly limited. Also, the final product of this technique is an enzyme-polymer complex which is soluble in organic solvents.
Entrapment of enzymes is described in U.S. Pat. No. 4,978,619. The products of this patent have an enzyme entrapped in gaps formed in a macromolecular gel matrix that is produced by dispersing the enzyme in the form of a fine powder and thus not solubilized as in the present invention described hereinafter in an organic solvent having dissolved therein a polymerizable monomer, polymerizing the monomer thereby giving rise to a gel matrix, and displacing the organic solvent in the gel matrix with an aqueous solvent. The method of this patent generates a polymer matrix containing hetero-geneous enzyme aggregates (i.e., a cluster of enzyme molecules).
The problems and limitations of the prior art are solved, avoided and/or reduced by the invention described herein.